Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.
Comp Biochem Physiol C Toxicol Pharmacol. 2011 Apr;153(3):269-79. doi: 10.1016/j.cbpc.2010.11.005. Epub 2010 Dec 4.
Our previous studies have provided evidence that birds have two isoforms of aryl hydrocarbon receptors (AHR1 and AHR2) and AHR nuclear translocators (ARNT1 and ARNT2) that potentially mediate toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. We have also shown that while both in vitro-expressed chicken AHR1 (ckAHR1) and AHR2 (ckAHR2) exhibit binding affinities to TCDD, only ckAHR1 but not ckAHR2 showed a TCDD-dose-dependent transactivation potency of chicken cytochrome P450 1A5 (ckCYP1A5) in in vitro reporter gene assays. To explore the molecular mechanism of functional difference in the two ckAHRs, the present study investigated the molecular characteristics and function of chicken ARNT (ckARNT) that is a potential dimerization partner for the activation of ckAHR. The full-length ckARNT1 and ckARNT2 cDNAs were isolated and their alternative splice variants were also identified. The ckARNT1 transcript was ubiquitously expressed in various tissues, but ckARNT2 showed restricted expressions in brain, kidney and eye, indicating a similar expression pattern to mammalian ARNTs. The expressions of tagged-ckARNT1 and -ckARNT2 were confirmed in a chicken hepatoma LMH cells by western blot analyses, and their interactions with each ckAHR and a specific recognition DNA element, xenobiotic response element (XRE), were examined by gel shift assays. The result showed that ckARNT1 and ckARNT2 dimerize with each ckAHR isoform and bind with the XRE in a TCDD-dependent manner. Hence, we conclude that functional loss on the dimerization with ckARNTs or the XRE binding is not the major cause of the deficient TCDD-dependency of ckAHR2 for the transactivation. Furthermore, in vitro reporter gene assays showed that transfected ckARNT1 failed to modulate the transcriptional induction of ckAHR-mediated ckCYP1A5 gene by TCDD in COS-7 and LMH cells, whereas ckARNT2 could potentiate the TCDD-dependent response in COS-7 but not in LMH cells. This suggests that ckARNT2 has a distinct role from ckARNT1 in AHR signaling pathway and in a cell-specific mode of action.
我们之前的研究已经证明鸟类有两种芳基烃受体（AHR1 和 AHR2）和 AHR 核转位蛋白（ARNT1 和 ARNT2），它们可能介导 2,3,7,8-四氯二苯并二恶英（TCDD）和相关化合物的毒性反应。我们还表明，尽管体外表达的鸡 AHR1（ckAHR1）和 AHR2（ckAHR2）都表现出对 TCDD 的结合亲和力，但只有 ckAHR1 而不是 ckAHR2 在体外报告基因检测中显示出 TCDD 剂量依赖性的鸡细胞色素 P450 1A5（ckCYP1A5）的转录激活能力。为了探讨这两种 ckAHR 功能差异的分子机制，本研究探讨了鸡 ARNT（ckARNT）的分子特征和功能，ckARNT 是激活 ckAHR 的潜在二聚化伴侣。分离了全长 ckARNT1 和 ckARNT2 cDNA，并鉴定了它们的可变剪接变体。ckARNT1 转录本在各种组织中广泛表达，但 ckARNT2 在脑、肾和眼中表达受限，表明其表达模式与哺乳动物 ARNTs 相似。通过 Western blot 分析证实了标记的 ckARNT1 和 -ckARNT2 在鸡肝癌 LMH 细胞中的表达，并通过凝胶迁移阻滞实验检测了它们与每个 ckAHR 和特定识别 DNA 元件（外源响应元件，XRE）的相互作用。结果表明，ckARNT1 和 ckARNT2 与每个 ckAHR 异构体二聚化，并以 TCDD 依赖的方式与 XRE 结合。因此，我们得出结论，与 ckARNTs 二聚化或 XRE 结合的功能丧失不是 ckAHR2 对 TCDD 依赖性转录激活缺陷的主要原因。此外，体外报告基因检测显示，在 COS-7 和 LMH 细胞中，转染的 ckARNT1 未能调节 TCDD 诱导的 ckAHR 介导的 ckCYP1A5 基因的转录诱导，而 ckARNT2 可增强 TCDD 依赖的反应，但在 LMH 细胞中不能。这表明 ckARNT2 在 AHR 信号通路中具有与 ckARNT1 不同的作用，并以细胞特异性的作用模式发挥作用。
